Phosphorylation of neurofilament proteins (NFPs) in vertebrate neurons regulates neurofilament assembly, transport and stability of large axonal structures. Phosphorylation of KSP (lys-ser-pro) repeat motifs in carboxy tail domains of high molecular weight neurofilament sub-units (NF-M and NF-H) contributes to increased axonal caliber, and, thereby, the conducting velocity of impulses in nerve fibers. In a normal neuron, phosphorylation of these KSP repeats seems to be topographically localized within the axonal compartment of neurons. Although all components of the cytoskeletal system of axons are synthesized in cell bodies, including the kinases involved in their phosphorylation, the phosphorylation of some elements such as neurofilaments occurs primarily in axons. The aberrant phosphorylation of the KSP repeats in perikarya has been proposed as a mechanism for pathological NF-accumulation in the cell bodies and proximal axons in several neurodegenerative diseases. These include Amyotrophic Lateral Sclerosis (ALS), Parkinson&#8217;s Disease, Dementia with Lewy bodies, diabetic neuropathy and Alzheimer's Disease with concomitant Lewy body pathology. Thus, phosphorylation of the NFs, and its regulation, seem to be involved in several physiological functions and pathological processes. However, the mechanisms responsible for the regulation of this phosphorylation are not well understood. The primary objective of this laboratory has been to understand the mechanism(s) of phosphorylation of NFs as well as other cytoskeletal proteins in neurons. This may provide insight into the processes underlying neurodegenerative disorders. Our previous studies have demonstrated that most ser/thr residues in the KSP repeats in human and rat NF-H are phosphorylated in vivo, suggesting that proline-directed kinases are responsible for their phosphorylation. In our recent studies we have shown that a family of mitogen activated kinases (Erk1/2, SAPK) and neuronal cyclin dependent kinase 5 (cdk5) can phosphorylate KSP repeats of NF-M and NF-H in their C-terminal tail domains in vitro and in vivo in neuronal and in transfected cells. For example, non-neuronal cell lines co-transfected with NF-M or NF-H, and kinases, that are either constituitively active or activated by a signal transduction pathway (MAP kinases), will induce robust phosphorylation of KSP repeats in neurofilament tail domains. These studies suggest that the activation of proline-directed kinase pathways in vivo are responsible for axonal NF-phosphorylation. We found that the selectivity of these kinases depends upon the nature of the KSP motifs. For example, in rat NF-H, where the majority of KSP repeats (repeating 41 times) are present as KSPXXXK motifs, are primarily phosphorylated by the activation of ERK1/2 kinases, while integrin a1/b1-mediated activation of cdk5 phosphorylation of human NF-H in a human cell line, has higher efficacy compared to activated ERK1/2. This is due to the fact that human NF-H has more KSPXK repeat motifs,, the consensus sequence for cdk5, repeating 32 times, than KSPXXXK (repeating 11 times). These studies suggest that different kinase cascades may mutually interact in the sequential phosphorylation of multiple sites in NFs, depending upon the species and environmental cues. A similar mechanism may be operative for other cytoskeletal proteins such as human tau; the microtubule associated protein found in axons. Numerous proline and non-proline-directed kinases have been shown to phosphorylate human tau in vivo and in vitro, producing hyperphosphorylated tau in the abnormal aggregates of paired helical filaments found in perikarya of Alzheimer neurons. Some clues as to the topographic regulation of phosphorylation in neurons have come from our summer studies on the squid giant axon at the Marine Biological Laboratory in Woods Hole. In axoplasm of the giant axon we have demonstrated active multimeric complexes of cytoskeletal proteins (tubulin and neurofilaments) associated with proline directed kinases such as cdc2-like kinases and Erk1/2 together with non-proline directed kinases such as CKI, PKA and CAMK. Such complexes are absent in the cell bodies of those axons; instead we found different, relatively inactive, complexes of cytoskeletal proteins and kinases. This suggests that the compartmentalization of cytoskeletal phosphorylation and its regulation depends on the nature of the &#8220;phosphorylating machines&#8221; that are segregated into the respective compartments. Significantly, we have followed up these studies by demonstrating that similar multimeric phosphorylating complexes can be extracted from rat brain lysates by identical techniques. We have continued our studies on neuronal cyclin dependent kinase 5 (cdk5) to further understand its role in nerve cell function and regulation. Our previous studies have shown that cdk5 null mice exhibit a unique phenotype characterized by perinatal mortality, disrupted cerebral cortical layering due to abnormal neuronal migration, lack of cerebellar foliation, and degeneration of neurons in the brain stem and spinal cord. Recently, in an attempt to rescue the cdk5 knockout phenotype, we have reconstituted cdk5 overexpression in a tissue specific manner in the brain in cdk5 null mice and examined its effect on the lethal phenotype. Unlike the cdk5 null mice, cdk5 that overexpress the cdk5 transgene with a p35 (the cyclin like cdk5 activator) promoter (TgKO mice) were viable and fertile. Since p35 is expressed primarily in the nervous system, in these mice cdk5 overexpression was limited to neurons. In contrast, wild type mice express cdk5 in neurons as well as astrocytes. The cerebral cortical layering pattern and cerebellar foliation was normal, and no degenerating neurons were seen in the brain stem and the spinal cord. We were thus able to reverse the phenotype and associated lethality of cdk5 null mice. This investigation confirms that cdk5 expression in the nervous system is critical for embryonic development and survival. In a separate study we have provided the evidence for a novel role for cross talk between cdk5 and MAP kinase pathway. Cdk5 down regulates the activity of MAP kinase kinase 1 (MEK1), the upstream regulator of ERK1 by phosphorylating its Thr-286 residue both in vitro and in vivo. Using p35 null mice which lack appreciable cdk5 activity, we show that MEK1 activity is regulated by cdk5 in vivo. These observations provide evidence for a cross talk between cdk5 and the MAP kinase pathway in the brain. This may serve as another regulatory mechanism that modulates signal transduction pathways during growth, differentiation and apoptotic cell death.